1. Field of the Invention
The present invention is in the fields of biochemistry and molecular biology. The invention relates to an assay for detecting nucleic acid cleavage reactions. More particularly, the invention relates to a continuous fluorometric assay for detecting nucleic acid cleavage reactions that are enzyme-mediated.
2. Description of the Related Art
Virtually all protocols in molecular biology require, at some point, cleavage of nucleic acids into smaller sized discrete fragments. In vitro cleavage of nucleic acids is typically accomplished with restriction endonucleases. Restriction endonucleases are commercially available enzymes, derived from bacteria, that recognize short DNA sequences and then cleave the double-stranded DNA at specific sites within, or adjacent to, the recognition sequence. These enzymes have been classified into three groups—Types I, II, and III. Type II restriction enzymes, which cleave a specific sequence of nucleotides and a separate methylase that modifies the same recognition sequence, are widely used in molecular cloning. A partial list of restriction enzymes and their recognition sequences is provided in Chapter 5 of Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (1989).
Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The cleavage sites provide specific landmarks for obtaining a physical map of DNA. Further, the ability to produce specific DNA fragments by cleavage with restriction enzymes makes it possible to purify these fragments by molecular cloning. In addition, restriction enzymes have been utilized extensively for finding restriction fragment length polymorphisms (RFLPs) in allelic genomic regions. The use of RFLPs as genetic markers has been exploited in genetic linkage analysis, determination of patterns of inheritance for genetic disease, mapping of genes to specific chromosomal loci, and genetic fingerprinting.
Many enzymes other than restriction endonucleases are routinely used in molecular cloning. For example, DNases, RNases, exonucleases, and helicases are utilized in molecular biology to effect strand separation or denaturation of nucleic acids. These enzymes are discussed generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (1989). Such enzymes are utilized in numerous processes in molecular biology that serve to amplify and detect DNA, such as, polymerase chain reaction (PCR) (described in U.S. Pat. Nos. 4,683,194, 4,683,195 and 4,683,202), ligase chain reaction (LCR) (described in published PCT application WO 89/09835), and catalytic hybridization amplification (CHA) (described in published PCT application WO 89/09284, and U.S. Pat. Nos. 5,011,769 and 4,876,187).
Ascertaining that nucleic acid cleavage has occurred, and evaluating the efficiency of the cleavage process, have traditionally been done using a gel electrophoresis assay system (Sambrook et al., supra). Such a system, however, is not only time-consuming and laborious, but the assay is discontinuous, meaning that the process cannot be monitored throughout the cleavage process. This is clearly a disadvantage in certain situations, such as where partial cleavage is desired, or where one needs to establish precise enzyme kinetic information. Further, the conventional assays are often inhibited by high concentrations of salt that may be required owing to the purification and solubility of the proteins involved. Finally, radioactive labeling of the substrates is often required to achieve the necessary level of sensitivity.
More recently, a continuous spectroscopic assay for endonucleases has been reported (Waters and Connolly, Anal. Biochem 204:204–209 (1992)). This assay is based on the hyperchromic effect resulting from turnover of a duplex oligonucleotide substrate to single-stranded DNA products. Although this technique is continuous, its scope is limited by its narrow dynamic range and limited range of substrate concentrations.
A sensitive non-isotopic enzyme linked immunoabsorbent assay (ELISA) for determining the DNA cleavage activity of restriction endonucleases was described by Jeltsch et al., Anal. Biochem. 213:234–240 (1993). This assay utilized DNA substrates that are labeled on both ends; one 5′ end is labeled with biotin, and the other 5′ end is labeled with fluorescein or digoxigenin. The use of biotin-labeled DNA in this assay renders the method discontinuous and necessitates extensive sample handling for the detection step.
Finally, endonuclease-catalyzed cleavage reactions of fluorophore-labeled oligonucleotides have been monitored by fluorescence resonance energy transfer (FRET) techniques (Ghosh et al., Nucleic Acids Res. 22:3155–3159 (1994)).
Fluorescence resonance energy transfer (FRET) (Forster, T., Ann. Phys. (Leipzig) 2:55–75 (1948); Stryer, L., Annu. Rev. Biochem. 47:819–846 (1978); Stryer, L., Proc. Natl. Acad. Sci. USA 58:719–726 (1967); Conrad and Brand, Biochemistry 7:777–787 (1968); Chen and Scott, Anal. Lett. 18:393 (1985); Wu and Brand, Anal. Biochem. 218:1–13 (1994)) is the transfer of electronic excitation energy by the Förster mechanism, and measures the distance between a pair of fluorophores (donor and acceptor) in macromolecules, in the range of 10–80 Angstroms (Å). Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790–8794 (1988), utilized FRET experiments to study the hybridization of complementary oligodeoxynucleotides. Upon hybridization, energy transfer was detected by both a decrease in fluorescein (donor) emission intensity and an enhancement of rhodamine (acceptor) emission. Cooper and Hagerman, Biochemistry 29:9261–9268 (1990), also utilized FRET to determine the interarm angles of a synthetic DNA four-way junction. However, these investigators reported that upon annealing of a fluorescent-modified strand and its unlabeled complementary strand, the probe fluorescence was quenched (Clegg et al, Biochemistry 31:4846–4856 (1992); Cooper and Hagerman, supra), and the wavelength of the emission spectrum was shifted upon the formation of duplex DNA. These results suggest that effects other than dipolar energy transfer mechanisms alter the donor fluorescence (in the presence or absence of acceptor at the ends of complementary strands), and that these effects must be examined in order to reliably measure distances in DNA molecules by FRET. Thus, the occurrence of nondipolar effects on fluorescently labeled DNA may distort the distances quantified by FRET in certain instances.
Thus, there exists a need in the art for a continuous, accurate, sensitive, and non-isotopic assay for detecting restriction enzyme mediated cleavage of nucleic acids.
Another class of enzymes that catalyze nucleic acid cleavage reactions are retroviral integrases. These enzymes are responsible for catalyzing the integration of viral DNA into the host organism's chromosomal DNA. Currently, the target of viral therapeutics is to screen compounds that inhibit these enzymes.
For example, one focus of AIDS research is to find specific inhibitors of each step in the replication cycle of the HIV retrovirus. Although progress has been made in targeting reverse transcription, parallel efforts in inhibiting other processes could lead to the development of new therapeutic agents. Retroviral integration is a particularly attractive target in the search for specific inhibitors due to the absence of any known cellular counterparts in the host. The combined use of antiviral drugs with different target specificities will facilitate the search for therapeutic intervention.
The currently established in vitro assay system for HIV DNA integration is based upon the detection of labeled 32P integrated products either by electrophoresis or by biotin-avidin interaction (the substrate DNA being radiolabeled with 32P at the 5′ end and biotin at the 3′ end). (Craigie et al., Nucleic Acids Res. 19:2729–2734(1991)). Unfortunately, these methods are time consuming and often do not yield precise kinetic information. A sensitive and more rapid assay is desired in order to screen vast numbers of potential drugs and natural products. A rapid, sensitive, and continuous in vitro assay for screening the vast number of potential enzyme inhibitors is clearly needed to provide precise kinetic information necessary to determine the relative effectiveness of any inhibitor. Also, an assay is needed that would not be affected by high salt concentrations.
Thus, in light of the foregoing, there exists a need in the art for a continuous assay for accurately and sensitively detecting enzyme-mediated nucleic acid cleavage in vitro.